1. Field of the Invention
The present invention relates to the rapid, continuous flow, processing of tissue for microscopic examination, from fixation to impregnation.
2. Description of the Related Art
Conventional methods prepare tissues for histology by incubation in separate solutions of phosphate-buffered 10% formaldehyde for fixation, a series of increasing concentrations of ethanol for dehydration, and xylene for clearing tissue of dehydration agent, prior to impregnation. Because of the time required for this process, usually 8 hours or longer, it is customary to complete these separate steps--fixation, dehydration, clearing, and impregnation--overnight in automated mechanical instruments designed for those tasks (see, for example, U.S. Pat. Nos. 3,892,197, 4,141,312, and 5,049,510). A typical automated tissue processor (TISSUE-TEK) requires more than eight hours and is programmed to process batches of tissue samples as follows.
Set Time Set Volume of Station Solution Concentration (min) Temperature P/V** Agitation Solution 1 Buffered 10% 50 40.degree. C. On On 2.2-3.2 Formalin liters 2 Buffered 10% 50 40.degree. C. On On 2.2-3.2 Formalin liters 3 Alcohol* 80% 50 40.degree. C. On On 2.2-3.2 L 4 Alcohol 95% 50 40.degree. C. On On 2.2-3.2 L 5 Alcohol 95% 50 40.degree. C. On On 2.2-3.2 L 6 Alcohol 100% 50 40.degree. C. On On 2.2-3.2 L 7 Alcohol 100% 50 40.degree. C. On On 2.2-3.2 L 8 Alcohol 100% 50 40.degree. C. On On 2.2-3.2 L 9 Xylene 100% 50 40.degree. C. On On 2.2-3.2 L 10 Xylene 100% 50 40.degree. C. On On 2.2-3.2 L 11 Paraffin 50 60.degree. C. On On 4 12 Paraffin 50 60.degree. C. On On 4 13 Paraffin 50 60.degree. C. On On 4 14 Paraffin 50 60.degree. C. On On 4 **pressure/vacuum cycle *the alcohol used in most laboratories is a mixture of 90% ethyl, 5% methyl and 5% isopropyl alcohol.
Such conventional methodology demands that the tissue specimens be sent from the operating room, medical office or other sites, to a pathology laboratory on one day; the tissue specimens be prepared overnight; and the pathologist render a diagnosis based on microscopic examination of tissue sections the next day at the earliest, almost 24 hours after delivery of the specimen to the laboratory (FIG. 1). In addition to the minimum one-day delay in giving a surgeon the benefit of a report from the pathologist, there are also problems associated with impeded work flow in the pathology laboratory necessitated by the requisite batch processing of specimens, the safety concerns that attended having instruments operating overnight, the risk of possible instrument failures and the need to monitor the instruments, and the waste of using large volumes of reagents for such processing when automated. Moreover, expensive measures are required to prevent exposure of laboratory personnel to fumes and toxic substances associated with the reagents used in this process. Also, the large volumes of solvent waste and paraffin debris produced by conventional methodology pollute the environment.
Conventional fixation and processing cause irreversible damage to the structure of DNA and particularly RNA that limits the application of genetic techniques for diagnosis and research. Consequently, most DNA and certainly RNA analysis require special precautions with handling of material, such as immediate freezing of fresh tissues, because retrospective genetic analysis is impaired by conventional tissue processing techniques.
Histological diagnosis of a frozen section suffers from multiple disadvantages in comparison to sections prepared from paraffin blocks: the slide prepared from a frozen section "does not possess . . . uniformity of quality"; "it is technically more difficult for serial sections of the same specimen to be examined"; "extreme caution must be exercised in cutting the specimen in order to ensure a sufficiently thin section and to avoid the possibility of damaging details of the specimen"; and all the slides must be prepared "while the tissue is in the initial frozen state" because, "[i]f the tissue is thawed and refrozen for sectioning, it is severely damaged" (U.S. Pat. No. 3,961,097).
There is an ever present interest in expediting tissue processing and analysis for diagnostic purposes. Furthermore, recent healthcare focus has been directed to lessening the cost of various procedures including tissue processing. The costs of tissue processing are related to time, the space required for preparation and analysis, reagents (both the amount required for processing and handling discard), and the number of personnel required. More importantly, patients and their physicians depend on evaluation and diagnosis by the pathologist to guide treatment. Reducing the amount of time needed to complete tissue processing would lessen the anxiety experienced during the period between obtaining the specimen and delivering the pathologist's report to the surgeon.
Others have recognized the need to shorten the time required for tissue processing, but they have made only modest improvements in the conventional methods. To accelerate tissue processing, U.S. Pat. Nos. 4,656,047, 4,839,194, and 5,244,787 use microwave energy; U.S. Pat. Nos. 3,961,097 and 5,089,288 use ultrasonic energy; and U.S. Pat. No. 5,023,187 uses infrared energy. U.S. Pat. No. 5,104,640 disclosed a non-aqueous composition of a fixative, a stabilizing agent, and a solubilizing agent that adheres a blood smear to a slide. However, the aforementioned patents do not teach or suggest that the entire process of preparing diagnostic tissue slides could be accomplished in less than two hours, starting from fixation and ending with impregnation, with continuous throughput of samples. The present invention provides such a process.